Cancer cells must avert apoptotic checkpoints to survive in unfavorable conditions, such as nutrient deprivation and hypoxia, which quickly lead to excessive demand on the protein folding capacity of the endoplasmic reticulum (ER). When the extent of unfolded proteins in the ER lumen reaches a critical level, the cell engages a set of evolutionarily conserved signal transduction pathways that are collectively known as the unfolded protein response (UPR). Major effectors of the UPR in mammalian cells are the ER-resident transmembrane sensors IRE11, PERK, and AFT6. These stress sensors initially expand the ER network, upregulate chaperones and arrest global translation to restore homeostasis. However, if the ER damage is severe, these ER resident stress sensors initiate apoptosis through poorly understood mechanisms. Sustained and high level ER stress is documented in many forms of cancer; hence, malignant cells must evolve mechanisms to evade the normally cytotoxic consequences of such stress. Efforts to restore the apoptotic outputs of the UPR hold promise as a therapeutic strategy to kill cancer cells. Excessive ER stress triggers the intrinsic apoptotic pathway, which is tightly regulated at the outer mitochondrial membrane by the pro-death BCL-2 family proteins BAX and BAK. However, the molecular chain of events leading from ER stress to mitochondrial BAX/BAK activation remains poorly understood. My laboratory has developed a process to purify the pre- mitochondrial apoptotic activity from the cytosolic extract of ER-stressed Bax-/-Bak-/- cells. Using this technology, we have identified two major apoptotic signals that converge on mitochondrial BAX/BAK. One signal is the BH3-only protein BID, which is cleaved into its shorter pro-apoptotic form by Caspase-2. We now seek to understand the events that lead from the sensing of misfolding proteins at the ER membrane to the catalytic activation of Caspase-2, one of the most poorly characterized mammalian caspases. From the active extract, we also recently purified a second novel component-an adaptor protein containing an SH2 domain and two SH3 domains, which our data suggest is a BID-independent apoptotic signal downstream of ER stress. We now aim to define the pro-apoptotic role of this adaptor protein in ER stress signaling. The long- term objectives of this proposal are to understand how cells detect ER stress, decide if the damage is lethal, and communicate this information to the cell death machinery, and to identify components in the pathway that can be manipulated to influence cell survival. Two specific aims are outlined: (1) Define the mechanism(s) by which ER stress activates Caspase-2, and (2) Determine the role of this SH2/SH3-containing adaptor protein in ER stress signaling. These studies will define the mechanisms that control apoptosis downstream of ER stress-a pathway that may represent a key therapeutic target in cancer cells.